Technology transfer of an oil-in-water vaccine-adjuvant for strengthening pandemic influenza preparedness in Indonesia.

With the current enzootic circulation of highly pathogenic avian influenza viruses, the ability to increase global pandemic influenza vaccine production capacity is of paramount importance. This has been highlighted by, and is one of the main pillars of, the WHO Global Action Plan for Influenza Vaccines (GAP). Such capacity expansion is especially relevant in developing countries. The Vaccine Formulation Laboratory at University of Lausanne is engaged in the technology transfer of an antigen-sparing oil-in-water adjuvant in order to empower developing countries vaccine manufacturers to increase pandemic influenza vaccine capacity. In a one-year project funded by United States Department of Health and Human Services, the Vaccine Formulation Laboratory transferred the process know-how and associated equipment for the pilot-scale manufacturing of an oil-in-water adjuvant to Bio Farma, Indonesia's state-owned vaccine manufacturer, for subsequent formulation with H5N1 pandemic influenza vaccines. This paper describes the experience acquired and lessons learnt from this technology transfer project.

Pathogen-induced hatching and population-specific life-history response to water-borne cues in brown trout (Salmo trutta)

Hatching is an important niche shift, and embryos in a wide range of taxa can either accelerate or delay this life-history switch in order to avoid stage-specific risks. Such behavior can occur in response to stress itself and to chemical cues that allow anticipation of stress. We studied the genetic organization of this phenotypic plasticity and tested whether there are differences among populations and across environments in order to learn more about the evolutionary potential of stress-induced hatching. As a study species, we chose the brown trout (Salmo trutta; Salmonidae). Gametes were collected from five natural populations (within one river network) and used for full-factorial in vitro fertilizations. The resulting embryos were either directly infected with Pseudomonas fluorescens or were exposed to waterborne cues from P. fluorescens-infected conspecifics. We found that direct inoculation with P. fluorescens increased embryonic mortality and induced hatching in all host populations. Exposure to waterborne cues revealed population-specific responses. We found significant additive genetic variation for hatching time, and genetic variation in trait plasticity. In conclusion, hatching is induced in response to infection and can be affected by waterborne cues of infection, but populations and families differ in their reaction to the latter.

Simple estimation of incident HIV infection rates in notification cohorts based on window periods of algorithms for evaluation of line-immunoassay result patterns.

BACKGROUND: Tests for recent infections (TRIs) are important for HIV surveillance. We have shown that a patient's antibody pattern in a confirmatory line immunoassay (Inno-Lia) also yields information on time since infection. We have published algorithms which, with a certain sensitivity and specificity, distinguish between incident (< = 12 months) and older infection. In order to use these algorithms like other TRIs, i.e., based on their windows, we now determined their window periods. METHODS: We classified Inno-Lia results of 527 treatment-naïve patients with HIV-1 infection < = 12 months according to incidence by 25 algorithms. The time after which all infections were ruled older, i.e. the algorithm's window, was determined by linear regression of the proportion ruled incident in dependence of time since infection. Window-based incident infection rates (IIR) were determined utilizing the relationship 'Prevalence = Incidence x Duration' in four annual cohorts of HIV-1 notifications. Results were compared to performance-based IIR also derived from Inno-Lia results, but utilizing the relationship 'incident = true incident + false incident' and also to the IIR derived from the BED incidence assay. RESULTS: Window periods varied between 45.8 and 130.1 days and correlated well with the algorithms' diagnostic sensitivity (R(2) = 0.962; P<0.0001). Among the 25 algorithms, the mean window-based IIR among the 748 notifications of 2005/06 was 0.457 compared to 0.453 obtained for performance-based IIR with a model not correcting for selection bias. Evaluation of BED results using a window of 153 days yielded an IIR of 0.669. Window-based IIR and performance-based IIR increased by 22.4% and respectively 30.6% in 2008, while 2009 and 2010 showed a return to baseline for both methods. CONCLUSIONS: IIR estimations by window- and performance-based evaluations of Inno-Lia algorithm results were similar and can be used together to assess IIR changes between annual HIV notification cohorts.

A double layer model of the gas bubble/water interface

Zeta potential is a physico-chemical parameter of particular importance to describe sorption of contaminants at the surface of gas bubbles. Nevertheless, the interpretation of electrophoretic mobilities of gas bubbles is complex. This is due to the specific behavior of the gas at interface and to the excess of electrical charge at interface, which is responsible for surface conductivity. We developed a surface complexation model based on the presence of negative surface sites because the balance of accepting and donating hydrogen bonds is broken at interface. By considering protons adsorbed on these sites followed by a diffuse layer, the electrical potential at the head-end of the diffuse layer is computed and considered to be equal to the zeta potential. The predicted zeta potential values are in very good agreement with the experimental data of H-2 bubbles for a broad range of pH and NaCl concentrations. This implies that the shear plane is located at the head-end of the diffuse layer, contradicting the assumption of the presence of a stagnant diffuse layer at the gas/water interface. Our model also successfully predicts the surface tension of air bubbles in a KCl solution. (c) 2012 Elsevier Inc. All rights reserved.

Determination of Transmembrane Water Fluxes in Neurons Elicited by Glutamate Ionotropic Receptors and by the Cotransporters KCC2 and NKCC1: A Digital Holographic Microscopy Study.

Digital holographic microscopy (DHM) is a noninvasive optical imaging technique that provides quantitative phase images of living cells. In a recent study, we showed that the quantitative monitoring of the phase signal by DHM was a simple label-free method to study the effects of glutamate on neuronal optical responses (Pavillon et al., 2010). Here, we refine these observations and show that glutamate produces the following three distinct optical responses in mouse primary cortical neurons in culture, predominantly mediated by NMDA receptors: biphasic, reversible decrease (RD) and irreversible decrease (ID) responses. The shape and amplitude of the optical signal were not associated with a particular cellular phenotype but reflected the physiopathological status of neurons linked to the degree of NMDA activity. Thus, the biphasic, RD, and ID responses indicated, respectively, a low-level, a high-level, and an "excitotoxic" level of NMDA activation. Moreover, furosemide and bumetanide, two inhibitors of sodium-coupled and/or potassium-coupled chloride movement strongly modified the phase shift, suggesting an involvement of two neuronal cotransporters, NKCC1 (Na-K-Cl) and KCC2 (K-Cl) in the genesis of the optical signal. This observation is of particular interest since it shows that DHM is the first imaging technique able to monitor dynamically and in situ the activity of these cotransporters during physiological and/or pathological neuronal conditions.

In vivo characterisation of a novel water-soluble Cyclosporine A prodrug for the treatment of dry eye disease.

Cyclosporine A (CsA) has been demonstrated to be effective for the treatment of a variety of ophthalmological conditions, including ocular surface disorders such as the dry eye disease (DED). Since CsA is characterised by its low water solubility, the development of a topical ophthalmic formulation represents an interesting pharmaceutical question. In the present study, two different strategies to address this challenge were studied and compared: (i) a water-soluble CsA prodrug formulated within an aqueous solution and (ii) a CsA oil-in-water emulsion (Restasis, Allergan Inc., Irvine, CA). First, the prodrug formulation was shown to have an excellent ocular tolerance as well as no influence on the basal tear production; maintaining the ocular surface properties remained unchanged. Then, in order to allow in vivo investigations, a specific analytical method based on ultra high pressure liquid chromatography coupled with triple quadrupole mass spectrometer (UHPLC-MS/MS) was developed and optimised to quantify CsA in ocular tissues and fluids. The CsA ocular kinetics in lachrymal fluid for both formulations were found to be similar between 15 min and 48 h. The CsA ocular distribution study evidenced the ability of the prodrug formulation to penetrate into the eye, achieving therapeutically active CsA levels in tissues of both the anterior and posterior segments. In addition, the detailed analysis of the in vivo data using a bicompartmental model pointed out a higher bioavailability and lower elimination rate for CsA when it is generated from the prodrug than after direct application as an emulsion. The interesting in vivo properties displayed by the prodrug solution make it a safe and suitable option for the treatment of DED.

Quantification of myocardial blood flow with 82Rb positron emission tomography: clinical validation with 15O-water.

PURPOSE: Quantification of myocardial blood flow (MBF) with generator-produced (82)Rb is an attractive alternative for centres without an on-site cyclotron. Our aim was to validate (82)Rb-measured MBF in relation to that measured using (15)O-water, as a tracer 100% of which can be extracted from the circulation even at high flow rates, in healthy control subject and patients with mild coronary artery disease (CAD). METHODS: MBF was measured at rest and during adenosine-induced hyperaemia with (82)Rb and (15)O-water PET in 33 participants (22 control subjects, aged 30 ± 13 years; 11 CAD patients without transmural infarction, aged 60 ± 13 years). A one-tissue compartment (82)Rb model with ventricular spillover correction was used. The (82)Rb flow-dependent extraction rate was derived from (15)O-water measurements in a subset of 11 control subjects. Myocardial flow reserve (MFR) was defined as the hyperaemic/rest MBF. Pearson's correlation r, Bland-Altman 95% limits of agreement (LoA), and Lin's concordance correlation ρ (c) (measuring both precision and accuracy) were used. RESULTS: Over the entire MBF range (0.66-4.7 ml/min/g), concordance was excellent for MBF (r = 0.90, [(82)Rb-(15)O-water] mean difference ± SD = 0.04 ± 0.66 ml/min/g, LoA = -1.26 to 1.33 ml/min/g, ρ(c) = 0.88) and MFR (range 1.79-5.81, r = 0.83, mean difference = 0.14 ± 0.58, LoA = -0.99 to 1.28, ρ(c) = 0.82). Hyperaemic MBF was reduced in CAD patients compared with the subset of 11 control subjects (2.53 ± 0.74 vs. 3.62 ± 0.68 ml/min/g, p = 0.002, for (15)O-water; 2.53 ± 1.01 vs. 3.82 ± 1.21 ml/min/g, p = 0.013, for (82)Rb) and this was paralleled by a lower MFR (2.65 ± 0.62 vs. 3.79 ± 0.98, p = 0.004, for (15)O-water; 2.85 ± 0.91 vs. 3.88 ± 0.91, p = 0.012, for (82)Rb). Myocardial perfusion was homogeneous in 1,114 of 1,122 segments (99.3%) and there were no differences in MBF among the coronary artery territories (p > 0.31). CONCLUSION: Quantification of MBF with (82)Rb with a newly derived correction for the nonlinear extraction function was validated against MBF measured using (15)O-water in control subjects and patients with mild CAD, where it was found to be accurate at high flow rates. (82)Rb-derived MBF estimates seem robust for clinical research, advancing a step further towards its implementation in clinical routine.

Chemistry, isotope values (δD, δ18O, δ34S(SO4)) and temperatures of the water inflows in two Gotthard tunnels, Swiss Alps

Water inflows in the Gotthard Highway Tunnel and in the Gotthard Exploration Tunnel are meteoric waters infiltrating at different elevations, on both sides of an important orographic divide. Limited interaction of meteoric waters with gneissic rocks produces Ca-HCO3 and Na-Ca-HCO3 waters, whereas prolonged interaction of meteoric waters with the same rocks generates Na-HCO3 to Na-SO4 waters. Waters circulating in Triassic carbonate-evaporite rocks have a Ca-SO4 composition. Calcium-Na-SO4 waters are also present. They can be produced through interaction of either Na-HCO3 waters with anhydrite or Ca-SO4 waters with a local gneissic rock, as suggested by reaction path modeling. An analogous simulation indicates that Na-HCO3 waters are generated through interaction of Ca-HCO3 waters with a local gneissic rock. The two main SO4-sources present in the Alps are leaching of upper Triassic sulfate minerals and oxidative dissolution of sulfide minerals of crystalline rocks. Values of delta S-34(SO4) < <similar to>+ 9 parts per thousand, are due to oxidative dissolution of sulfide minerals, whereas delta S-34(SO4) > similar to+ 9 parts per thousand are controlled either by bacterial SO4 reduction or leaching of upper Triassic sulfate minerals. Most waters have temperatures similar to the expected values for a geothermal gradient of 22 degreesC/km and are close to thermal equilibrium with rocks. However relatively large, descending flows of cold waters and ascending flows of warm waters are present in both tunnels and determine substantial cooling and heating, respectively, of the interacting rocks. The most import upflow zone of warm, Na-rich waters is below Guspisbach, in the Gotthard Highway Tunnel, at 6.2-9.0 km from the southern portal. These warm waters have equilibrium temperatures of 65-75 degreesC and therefore constitute an important low-enthalpy geothermal resource. (C) 2001 Elsevier Science Ltd. All rights reserved.

Fat signal suppression for coronary MRA at 3T using a water-selective adiabatic T2 -preparation technique.

PURPOSE: To improve fat saturation in coronary MRA at 3T by using a spectrally selective adiabatic T2 -Prep (WSA-T2 -Prep). METHODS: A conventional adiabatic T2 -Prep (CA-T2 -Prep) was modified, such that the excitation and restoration pulses were of differing bandwidths. On-resonance spins are T2 -Prepared, whereas off-resonance spins, such as fat, are spoiled. This approach was combined with a CHEmically Selective Saturation (CHESS) pulse to achieve even greater fat suppression. Numerical simulations were followed by phantom validation and in vivo coronary MRA. RESULTS: Numerical simulations demonstrated that augmenting a CHESS pulse with a WSA-T2 -Prep improved robustness to B1 inhomogeneities and that this combined fat suppression was effective over a broader spectral range than that of a CHESS pulse in a conventional T2 -Prepared sequence. Phantom studies also demonstrated that the WSA-T2 -Prep+CHESS combination produced greater fat suppression across a range of B1 values than did a CA-T2 -Prep+CHESS combination. Lastly, in vivo measurements demonstrated that the contrast-to-noise ratio between blood and myocardium was not adversely affected by using a WSA-T2 -Prep, despite the improved abdominal and epicardial fat suppression. Additionally, vessel sharpness improved. CONCLUSION: The proposed WSA-T2 -Prep method was shown to improve fat suppression and vessel sharpness as compared to a CA-T2 -Prep technique, and to also increase fat suppression when combined with a CHESS pulse.

Water chemistry and isotope composition of the Acquarossa thermal system, Ticino, Switzerland

The thermal springs of Acquarossa and the nearby mineral springs of Soia have outlet temperatures of 12 degrees to 25 degrees C, TDS of 2290 to 3000 mg/kg and Ca-SO4 to Ca-SO4-HCO3 composition. Chemical geothermometers suggest reservoir temperatures close to 60 degrees C. P-CO2 values at depth are estimated to range from 0.3 to 2 bar. delta D and delta(18)O values indicate a meteoric origin and recharge elevations of 1600 +/- 150 m above sea level (a.s.l.) for these thermal and mineral waters. All these waters discharge from the overturned limb of the Simano nappe, probably dose to the contact between basement and underlying cover rocks. They therefore represent rain waters that descend slowly, heat at depth and locally rise relatively quickly to the surface, preserving part of their physical and chemical characteristics. (C) 1999 CNR. Published by Elsevier Science Ltd. All rights reserved.

Evaporation from a shallow water table: Diurnal dynamics of measured and simulated water and heat regime at the vicinity of a drying sand surface

Accurate estimates of water losses by evaporation from shallow water tables are important for hydrological, agricultural, and climatic purposes. An experiment was conducted in a weighing lysimeter to characterize the diurnal dynamics of evaporation under natural conditions. Sampling revealed a completely dry surface sand layer after 5 days of evaporation. Its thickness was <1 cm early in the morning, increasing to reach 4?5 cm in the evening. This evidence points out fundamental limitations of the approaches that assume hydraulic connectivity from the water table up to the surface, as well as those that suppose monotonic drying when unsteady conditions prevail. The computed vapor phase diffusion rates from the apparent drying front based on Fick's law failed to reproduce the measured cumulative evaporation during the sampling day. We propose that two processes rule natural evaporation resulting from daily fluctuations of climatic variables: (i) evaporation of water, stored during nighttime due to redistribution and vapor condensation, directly into the atmosphere from the soil surface during the early morning hours, that could be simulated using a mass transfer approach and (ii) subsurface evaporation limited by Fickian diffusion, afterward. For the conditions prevailing during the sampling day, the amount of water stored at the vicinity of the soil surface was 0.3 mm and was depleted before 11:00. Combining evaporation from the surface before 11:00 and subsurface evaporation limited by Fickian diffusion after that time, the agreement between the estimated and measured cumulative evaporation was significantly improved.

Increased therapeutic window for the R91W mutant form of Rpe65 compared to Rpe65 null backgroud

Purpose:Given the advances of gene therapy studies to cure RPE65-derived Leber Congenital Amaurosis (LCA) (clinical trials phase I) and the heterogeneity of the targeted patients both genetically and phenotypically, it is of prime importance to examine the rescue efficiency of gene transfer in different mutant contexts. Indeed, half of these mutations are missense mutations, leading to potential residual RPE65 activity. Consequently, we wanted to evaluate the effect on retinal activity and cone survival of lentivirus-mediated gene therapy in the R91W knock-in mouse model expressing the mutant Rpe65R91W gene (Samardzija et al. 2008), a mutation found in LCA patients. Notably we investigated whether if the therapeutic window is prolonged in comparison to null mutations. Methods:An HIV-1-derived lentiviral vector (LV) expressing either the GFP or the mouse Rpe65 cDNA under the control of a 0.8 kb fragment of the human Rpe65 promoter (R0.8) was produced by transient transfection of 293T cells. LV-R0.8-RPE65 or GFP was injected into 5-days-old (P5) or 1 month-old R91W mice. Functional rescue was assessed by ERG (1 and 4 months post-injection) and pupillary light response (PLR) recordings and cone survival by histological analysis. Results:Increased light sensitivity was detected by scotopic ERG in animals injected with LV-R0.8-RPE65 at both P5 and 1 month compared to GFP-treated animals or untreated mice. PLR was also improved in some eyes and histological analysis of cone markers showed that the density of cones reached the wild type level in the region of wt RPE65 delivery after treatment at P5. However, the rescue effect of the injection at 1 month was limited and attained 60% of the wild type level, but still more cones were observed in the treated area than in 1 month-old untreated Rpe65R91W mice. Conclusions:We were able to show that lentivirus-mediated Rpe65 gene transfer not only increases retinal activity of the Rpe65R91W mouse and survival of cones after treatment at P5 but also after treatment at 1 month. However even if the treatment at 1 month is more limited (60% of the wild type level) than treatment at P5, the amount of cone markers is increased compared to the proportion found at 1 month of age in untreated animals. This results contrast with the lack of cone rescue by treatment at 1 month of age in Rpe65-/- (Bemelmans et al, 2006). Thus patient suffering from R91W mutation might benefit from a prolonged therapeutic window.

Under- and over-water halves of Gyrinidae beetle eyes harbor different corneal nanocoatings providing adaptation to the water and air environments.

Whirligig beetles (Gyrinidae) inhabit water surfaces and possess unique eyes which are split into the overwater and underwater parts. In this study we analyze the micro- and nanostructure of the split eyes of two Gyrinidae beetles genera, Gyrinus and Orectochilus. We find that corneae of the overwater ommatidia are covered with maze-like nanostructures, while the corneal surface of the underwater eyes is smooth. We further show that the overwater nanostructures possess no anti-wetting, but the anti-reflective properties with the spectral preference in the range of 450-600 nm. These findings illustrate the adaptation of the corneal nanocoating of the two halves of an insect's eye to two different environments. The novel natural anti-reflective nanocoating we describe may find future technological applications.

Positive contrast visualization of iron oxide-labeled stem cells using inversion-recovery with ON-resonant water suppression (IRON)

In proton magnetic resonance imaging (MRI) metallic substances lead to magnetic field distortions that often result in signal voids in the adjacent anatomic structures. Thus, metallic objects and superparamagnetic iron oxide (SPIO)-labeled cells appear as hypointense artifacts that obscure the underlying anatomy. The ability to illuminate these structures with positive contrast would enhance noninvasive MR tracking of cellular therapeutics. Therefore, an MRI methodology that selectively highlights areas of metallic objects has been developed. Inversion-recovery with ON-resonant water suppression (IRON) employs inversion of the magnetization in conjunction with a spectrally-selective on-resonant saturation prepulse. If imaging is performed after these prepulses, positive signal is obtained from off-resonant protons in close proximity to the metallic objects. The first successful use of IRON to produce positive contrast in areas of metallic spheres and SPIO-labeled stem cells in vitro and in vivo is presented.